Immunoconjugates with an intracellularly-cleavable linkage

ABSTRACT

The present invention relates to therapeutic conjugates with improved ability to target various diseased cells containing a targeting moiety (such as an antibody or antibody fragment), a linker and a therapeutic moiety, and further relates to processes for making and using the conjugates.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/267,910, filed Sep. 16, 2016, which was a divisional of U.S. patentapplication Ser. No. 15/133,675 (how issued U.S. Pat. No. 9,480,756),filed Apr. 20, 2016, which was a divisional of U.S. patent applicationSer. No. 14/636,790 (now issued U.S. Pat. No. 9,345,783), filed Mar. 3,2015, which was a divisional of U.S. patent application Ser. No.14/294,651 (now issued U.S. Pat. No. 8,999,344), filed Jun. 3, 2014,which was a divisional of U.S. patent application Ser. No. 14/048,222(now issued U.S. Pat. No. 9,102,735), filed Oct. 8, 2013, which was adivisional of U.S. patent application Ser. No. 13/705,756 (now issuedU.S. Pat. No. 8,759,496), filed Dec. 5, 2012, which was a divisional ofU.S. patent application Ser. No. 13/291,238 (now issued U.S. Pat. No.8,741,300), filed Nov. 8, 2011, which was a divisional of U.S. patentapplication Ser. No. 13/164,275 (now issued U.S. Pat. No. 8,080,250),filed Jun. 20, 2011, which was a divisional of U.S. patent applicationSer. No. 12/629,404 (now issued U.S. Pat. No. 7,999,083), filed Dec. 2,2009, which claimed the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application 61/207,890, filed Feb. 13, 2009. The textof each of the priority applications is incorporated herein by referencein its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NumberCA114802 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to therapeutic conjugates with improvedability to target various cancer cells, infectious disease organismsand/or for treating autoimmune diseases, which conjugates contain atargeting (binding) moiety and a therapeutic moiety belonging to thecamptothecin group of drugs. The targeting and therapeutic moieties arelinked via an intracellularly cleavable linkage that increasestherapeutic efficacy.

BACKGROUND OF THE INVENTION

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer, and virtually noapplication in other diseases, such as infectious and autoimmunediseases. The toxic agent is most commonly a chemotherapeutic drug,although particle-emitting radionuclides, or bacterial or plant toxinshave also been conjugated to MAbs, especially for the therapy of cancer(Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-243) and, more recently, with radioimmunoconjugates for thepreclinical therapy of certain infectious diseases (Dadachova andCasadevall, Q J Nucl Med Mol Imaging 2006; 50(3):193-204; incorporatedherein by reference).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using very welldefined conjugation chemistries, often at specific sites remote from theMAbs antigen binding regions; (c) MAb-chemotherapeutic drug conjugatescan be made more reproducibly than chemical conjugates involving MAbsand bacterial or plant toxins, and as such are more amenable tocommercial development and regulatory approval; and (d) theMAb-chemotherapeutic drug conjugates are orders of magnitude less toxicsystemically than radionuclide MAb conjugates.

Early work on protein-drug conjugates indicated that a drug preferablyis released in its original form, once it has been internalized into atarget cell, for the protein-chemotherapeutic drug conjugate to be auseful therapeutic. Trouet et al. (Proc. Natl. Acad. Sci. USA 79:626-629(1982)) showed the advantage of using specific peptide linkers, betweenthe drug and the targeting moiety, which are cleaved lysosomally toliberate the intact drug. Notably, MAb-chemotherapeutic drug conjugatesprepared using mild acid-cleavable linkers, such as those containing ahydrazone, were developed, based on the observation that the pH insidetumors was often lower than normal physiological pH (Willner et al.,U.S. Pat. No. 5,708,146; Trail et al. (Science 261:212-215 (1993)). Thefirst approved MAb-drug conjugate, Gemtuzumab Ozogamicin, incorporates asimilar acid-labile hydrazone bond between an anti-CD33 antibody,humanized P67.6, and a potent calicheamicin derivative. Sievers et al.,J Clin Oncol. 19:3244-3254 (2001); Hamann et al., Bioconjugate Chem. 13:47-58 (2002). In some cases, the MAb-chemotherapeutic drug conjugateswere made with reductively labile hindered disulfide bonds between thechemotherapeutic drugs and the MAb (Liu et al., Proc Natl Acad Sci USA93: 8618-8623 (1996)).

Yet another cleavable linker involves cathepsin B-labile dipeptidespacers, such as Phe-Lys or Val-Cit, similar to the lysosomally labilepeptide spacers of Trouet et al. containing from one to four aminoacids, which additionally incorporated a collapsible spacer between thedrug and the dipeptide (Dubowchik, et al., Bioconjugate Chem. 13:855-869(2002); Firestone et al., U.S. Pat. No. 6,214,345 B1; Doronina et al.,Nat Biotechnol. 21: 778-784 (2003)). The latter approaches were alsoutilized in the preparation of an immunoconjugate of camptothecin(Walker et al., Bioorg Med Chem Lett. 12:217-219 (2002)). Anothercleavable moiety that has been explored is an ester linkage incorporatedinto the linker between the antibody and the chemotherapeutic drug.Gillimard and Saragovi have found that when an ester of paclitaxel wasconjugated to anti-rat p75 MAb, MC192, or anti-human TrkA MAb, 5C3, theconjugate was found to exhibit target-specific toxicity. Gillimard andSaragovi, Cancer Res. 61:694-699 (2001).

The conjugates of the instant invention possess greater efficacy, inmany cases, than unconjugated or “naked” antibodies or antibodyfragments, although such unconjugated targeting molecules have been ofuse in specific situations. In cancer, for example, naked antibodieshave come to play a role in the treatment of lymphomas (alemtuzumab andrituxumab), colorectal and other cancers (cetuximab and bevacizumab),breast cancer (trastuzumab), as well as a large number now in clinicaldevelopment (e.g., epratuzumab). In most of these cases, clinical usehas involved combining these naked, or unconjugated, antibodies withother therapies, such as chemotherapy or radiation therapy.

A variety of antibodies are also in use for the treatment of autoimmuneand other immune dysregulatory diseases, such as tumor necrosis factor(TNF) and B-cell (rituxumab) antibodies in arthritis, and are beinginvestigated in other such diseases, such as the B-cell antibodies,rituxumab and epratuzumab, in systemic lupus erythematosus and Sjögren'ssyndrome, as well as juvenile diabetes and multiple sclerosis. Nakedantibodies are also being studied in sepsis and septic shock,Alzheimer's disease, and infectious diseases. The development ofanti-infective monoclonal antibodies has been reviewed recently byReichert and Dewitz (Nat Rev Drug Discovery 2006; 5:191-195),incorporated herein by reference, which summarizes the prioritypathogens against which naked antibody therapy has been pursued,resulting in only 2 pathogens against which antibodies are either inPhase III clinical trials or are being marketed (respiratory syncytialvirus and methicillin-resistant Staphylococcus aureus), with 25 othersin clinical studies and 20 discontinued during clinical study.

There is a need to develop more potent anti-pathogen or anti-cancerantibodies and other binding moieties. Such antibody-mediatedtherapeutics can be developed for the treatment of many differentpathogens, including bacteria, fungi, viruses, and parasites, either asnaked (unconjugated), radiolabeled, or drug/toxin conjugates. There is afurther need to develop more effective antibody conjugates withintracellularly cleavable linkers, of use for the treatment of cancer,pathogens and other diseases. In the case of delivering drug/toxin orradionuclide conjugates, this can be accomplished by direct antibodyconjugation or by indirect methods, referred to as pretargeting, where abispecific antibody is used to target to the lesion, while thetherapeutic agent is secondarily targeted by binding to one of the armsof the bispecific antibody that has localized at the site of thepathogen, the cancer or whatever lesion is being treated (Goldenberg etal., J Clin Oncol. 2006 Feb. 10; 24(5):823-34.; Goldenberg et al., JNucl Med. 2008 January; 49(1):158-63, each incorporated herein byreference in their entirety).

SUMMARY OF THE INVENTION

The present invention resolves an unfulfilled need in the art byproviding improved methods and compositions for preparation ofdrug-binding moiety conjugates. The disclosed methods and compositionsare of use for the treatment of a variety of diseases and conditionswhich are refractory or less responsive to other forms of therapy, andcan include diseases against which suitable targeting (binding) moietiesfor selective targeting can be developed, or are available or known.Preferably, the targeting moiety is an antibody, antibody fragment,bispecific or other multivalent antibody, or other antibody-basedmolecule or compound. The antibody can be of various isotypes,preferably IgG1, IgG2a, IgG3, IgG4 or IgA, and can be a chimerichuman-mouse, a chimeric human-primate, a humanized (human framework andmurine hypervariable (CDR) regions), or fully human antibody, as well asvariations thereof, such as half-IgG4 antibodies (referred to as“unibodies”) as described by van der Neut Kolfschoten et al. (Science2007; 317:1554-1557), incorporated herein by reference. However, otherbinding moieties known in the art, such as aptamers, avimers ortargeting peptides, may be used. Preferred diseases or conditionsagainst which such targeting moieties exist are, for example, cancer,immune dysregulatory conditions, including autoimmune diseases andinflammatory diseases, and diseases caused by infectious organisms.

The disclosed methods and compositions may thus be applied for treatmentof diseases and conditions for which targeting moieties are of use todeliver cytotoxic agents. Such diseases or conditions may becharacterized by the presence of a target molecule or target cell thatis insufficiently affected when unconjugated, or naked, targetingmoieties are used, such as in the immunotherapy of cancer or ofinfection with pathogenic organisms. (For methods of makingimmunoconjugates of antibodies with isotopes, drugs, and toxins for usein disease therapies, see, e.g., U.S. Pat. Nos. 4,699,784; 4,824,659;5,525,338; 5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284;6,306,393; 6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856;7,259,240 and U.S. Patent Appln. Publ. Nos. 20050175582 (now abandoned);20050136001; 20040166115 (now abandoned); 20040043030 (now abandoned);20030068322 (now abandoned) and 20030026764 (now abandoned), theExamples section of each incorporated herein by reference.)

In a preferred embodiment, camptothecin (CPT) and its analogs andderivatives are preferred chemotherapeutic moieties, although theinvention is not so limited. Other chemotherapeutic moieties that arewithin the scope of the invention are taxanes (e.g, baccatin III,taxol), epothilones, anthracycline drugs (e.g., doxorubicin (DOX),epirubicin, morpholinodoxorubicin (morpholino-DOX),cyanomorpholino-doxorubicin (cyanomorpholino-DOX), and2-pyrrolinodoxorubicin (2-PDOX); see Priebe W (ed.), ACS symposiumseries 574, published by American Chemical Society, Washington D.C.,1995 (332pp) and Nagy et al., Proc. Natl. Acad. Sci. USA 93:2464-2469,1996), benzoquinoid ansamycins exemplified by geldanamycin (DeBoer etal., Journal of Antibiotics 23:442-447, 1970; Neckers et al., Invest.New Drugs 17:361-373, 1999), and the like. Preferably, in theimmunoconjugates of the preferred embodiments of the present invention,the targeting moiety links to at least one chemotherapeutic moiety;preferably 1 to about 5 chemotherapeutic moieties; most preferably about7 to about 12 chemotherapeutic moieties.

With regard to the CPT group of drugs, issues of insolubility in aqueousbuffers and the lability of the δ-lactone moiety of the E-ring of theirstructures under physiological conditions are relevant. One approach hasbeen to acylate the 20-hydroxyl group with an amino acid, and couple theα-amino group of the amino acid to poly-L-glutamic acid (Singer et al.in The Camptothecins: Unfolding Their Anticancer Potential, Liehr J. G.,Giovanella, B. C. and Verschraegen (eds), NY Acad Sci., NY 922:136-150(2000)). This approach relies on the passive diffusion of a polymericmolecule into tumor sites. This glycine conjugation has also beenreported as a method of making a water-soluble derivative of CPT(Vishnuvajjala et al., U.S. Pat. No. 4,943,579) and in the preparationof a PEG-derivatized CPT (Greenwald, et al. J. Med. Chem. 39: 1938-1940(1996)). In the latter case, the approach has been devised in thecontext of developing water-soluble and long acting forms of CPT,whereby CPT's in vivo half-life is enhanced, and the drug is graduallyreleased from its conjugate while in circulation in vivo. An example ofa water soluble CPT derivative is CPT-11. Extensive clinical data areavailable concerning CPT-11's pharmacology and its in vivo conversion tothe active SN-38 (Iyer and Ratain, Cancer Chemother Pharmacol. 42:S31-43(1998); Mathijssen et al., Clin Cancer Res. 7:2182-2194 (2002); Rivory,Ann NY Acad Sci. 922:205-215, 2000)). The active form SN-38 is about 2to 3 orders of magnitude more potent than CPT-11.

In certain exemplary embodiments, drug conjugates of antibodies orantibody fragments may be used for targeting the therapeutic drug topathogens, such as bacteria, viruses, fungi, and parasites. In preferredembodiments, such drug-conjugated targeting moieties can be used incombination with other therapeutic modalities, such as anti-fungal,antibiotics and anti-viral drugs and/or naked antibodies,immunomodulators (e.g., interferons, interleukins, and/or cytokines).The use of radioimmunotherapy for the treatment of infectious organismsis disclosed, for example, in U.S. Pat. Nos. 4,925,648; 5,332,567;5,439,665; 5,601,825; 5,609,846; 5,612,016; 6,120,768; 6,319,500;6,458,933; 6,548,275; and in U.S. Patent Application Publication Nos.20020136690 and 20030103982, the Examples section of each of which isincorporated herein by reference.

In certain embodiments involving treatment of cancer, the drugconjugates may be used in combination with surgery, radiation therapy,chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy,immunomodulators, vaccines, and the like. Similar combinations arepreferred in the treatment of other diseases amenable to targetingmoieties, such as autoimmune diseases. For example, camptothecinconjugates can be combined with TNF inhibitors, B-cell antibodies,interferons, interleukins, and other effective agents for the treatmentof autoimmune diseases, such as rheumatoid arthritis, systemic lupuserythematosis, Sjögren's syndrome, multiple sclerosis, vasculitis, aswell as type-I diabetes (juvenile diabetes). These combination therapiescan allow lower doses of each therapeutic to be given in suchcombinations, thus reducing certain severe side effects, and potentiallyreducing the courses of therapy required. In viral diseases, the drugimmunoconjugates can be combined with other therapeutic drugs,immunomodulators, naked MAbs, or vaccines (e.g., MAbs against hepatitis,HIV, or papilloma viruses, or vaccines based on immunogens of theseviruses). Antibodies and antigen-based vaccines against these and otherviral pathogens are known in the art and, in some cases, already incommercial use.

In one embodiment, the invention relates to a process of preparingconjugates, wherein a drug is first derivatized with a first linker,which first linker contains a reactive moiety that is capable ofcombining with a second linker that additionally contains atargeting-moiety-coupling group; wherein the first linker also possessesa defined polyethylene glycol (PEG) moiety for water-solubility, andoptionally an intracellularly-cleavable moiety cleavable byintracellular peptidases or cleavable by the low pH environment ofendosomal and lysosomal vescicles, and optionally an amino acid spacerbetween the drug and the first linker; wherein the second linkercontains a reactive group capable of reacting with drug-(first linker)conjugate by the copper (+1) ion-catalyzed acetylene-azide cycloadditionreaction, referred to as ‘click chemistry’. Preferably, the defined PEGmoiety is a low molecular weight PEG with a defined number of monomericsubunits, as discussed below.

Another embodiment relates to a process of preparing conjugates asdiscussed in the paragraph above, wherein the second linker has a singletargeting-moiety-coupling group, but multiples of the reactive groupcapable of reacting with drug-(first linker) conjugate, therebyamplifying the number of drug molecules conjugated to the targetingmoiety.

A further embodiment relates to a process of preparing conjugates,wherein the linker is first conjugated to a drug, thereby producing adrug-linker conjugate; wherein said drug-linker conjugate preparationinvolves the selective protection and deprotection of a more reactivegroup in a drug containing multiple functional groups; wherein saiddrug-linker conjugate is optionally not purified; and wherein saiddrug-linker conjugate is subsequently conjugated to a monoclonalantibody or fragment.

Yet another embodiment is a method of treating cancer (malignancy), anautoimmune disease, an infection, or an infectious lesion with theconjugates described herein. Alternative embodiments concern thedrug-targeting moiety conjugates made by the claimed processes and/orkits for performing the claimed processes.

In one embodiment, the invention relates to an immunoconjugatecomprising:

(a) a targeting moiety;

(b) a chemotherapeutic moiety; and

(c) a linker covalently attached to the targeting moiety via a targetingmoiety-binding group and to the chemotherapeutic moiety via anintracellularly-cleavable moiety. In another embodiment, the inventionrelates to an immunoconjugate comprising:

(a) targeting moiety;

(b) a chemotherapeutic moiety; and

(c) a linker covalently attached to the targeting moiety via a targetingmoiety-binding group and to the chemotherapeutic moiety via anintracellularly-cleavable moiety; wherein said linker attachment totherapeutic moiety further comprises an L-amino acid or a polypeptidemade up of up to four L-amino acids.

In one embodiment, the intracellularly-cleavable moiety is a carbonatecomprising an activated hydroxyl group of the chemotherapeutic moietyand a substituted ethanolamine moiety or a 4-aminobenzyl alcohol, andthe latter is attached, via its amino group, to a cross-linkerterminating in the targeting moiety-binding group; and wherein thesubstituted ethanolamine moiety is derived from a natural L amino acid,with the carboxylic acid group of the latter replaced with ahydroxymethyl moiety; and wherein the 4-aminobenzyl alcohol isoptionally substituted with a C₁-C₁₀ alkyl group at the benzylicposition.

In a preferred embodiment, the intracellularly-cleavable moiety is acarbonate comprising an activated hydroxyl group of the chemotherapeuticmoiety and a substituted ethanolamine moiety, and the latter, via itsamino group, is attached to an L-amino acid or a polypeptide comprisingup to four L-amino acid moieties; wherein the N-terminus is attached toa cross-linker terminating in the targeting moiety-binding group; andwherein the substituted ethanolamine moiety is optionally derived froman L amino acid, with the carboxylic acid group of the latter replacedwith a hydroxymethyl moiety.

In another preferred embodiment, the intracellularly-cleavable moiety isa carbonate comprising an activated hydroxyl group of thechemotherapeutic moiety and a 4-aminobenzyl alcohol or substituted4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkyl group at thebenzylic position, and the latter, via its amino group, is attached toan L-amino acid or a polypeptide comprising up to four L-amino acidmoieties; wherein the N-terminus is attached to a cross-linkerterminating in the targeting moiety-binding group.

In certain embodiments, an amino group of a chemotherapeutic moiety iscoupled to the activated hydroxyl group of a substituted, andamine-protected, ethanolamine moiety or a 4-aminobenzyl alcohol, and thelatter is attached, via its amino group, to an L-amino acid or apolypeptide comprising up to four L-amino acid moieties; wherein theN-terminus is attached to a cross-linker terminating in the targetingmoiety-binding group; wherein said substituted ethanolamine moiety isoptionally derived from an L amino acid, with the carboxylic acid groupof the latter replaced with a hydroxymethyl moiety; and wherein the4-aminobenzyl alcohol is optionally substituted with a C₁-C₁₀ alkylgroup at the benzylic position. The bifunctional drug derivative is thenconjugated to a targeting moiety to obtain an immunoconjugate asdiscussed above. Upon targeting the disease site with theimmunoconjugate, the immunoconjugate is endocytosed and catabolized torelease the drug-linker moiety; wherein the free amino group of thesubstituted ethanolamine moiety assists in the liberation of free drugby nucleophilic attack at the carbonyl group of the carbamate moiety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Preclinical in vivo therapy of athymic nude mice, bearing Capan1 human pancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 2. Preclinical in vivo therapy of athymic nude mice, bearing BxPC3human pancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 3. Preclinical in vivo therapy of athymic nude mice, bearing LS174Thuman colon carcinoma, with hMN-14-CL2A-SN-38 conjugate.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

The term targeting moiety as used herein refers to a molecule, complexor aggregate, that binds specifically or selectively to a targetmolecule, cell, particle, tissue or aggregate. The skilled artisan willunderstand that specific binding refers to binding to a particulartarget without cross-reactivity to other targets, while selectivebinding refers to preferential binding to a particular target. Inpreferred embodiments, a targeting moiety is an antibody, antibodyfragment, bispecific antibody or other antibody-based molecule orcompound. However, other examples of targeting moieties are known in theart and may be used, such as aptamers, avimers, receptor-bindingligands, nucleic acids, biotin-avidin binding pairs, binding peptides orproteins, etc. The terms “targeting moiety” and “binding moiety” areused synonymously herein.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2a, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317(14September):1554-1557). Regardless of structure, an antibody fragment ofuse binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains, recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”), and minimal recognition units consisting of the aminoacid residues that mimic the hypervariable region, such as CDRs. The Fvfragments may be constructed in different ways to yield multivalentand/or multispecific binding forms. In the case of multivalent, theyhave more than one binding site against the specific epitope, whereaswith multispecific forms, more than one epitope (either of the sameantigen or against one antigen and a different antigen) is bound. Asused herein, the term antibody component includes an entire antibody, afusion protein, and fragments thereof.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector or immunological functions, such ascomplement fixation and ADCC (antibody-dependent cell cytotoxicity),which set mechanisms into action that may result in cell lysis. However,the Fc portion may not be required for therapeutic function of theantibody, but rather other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,such as inhibition of heterotypic or homotypic adhesion, andinterference in signaling pathways, may come into play and interferewith disease progression. Naked antibodies include both polyclonal andmonoclonal antibodies, and fragments thereof, that include murineantibodies, as well as certain recombinant antibodies, such as chimeric,humanized or human antibodies and fragments thereof. Therefore, in somecases a “naked antibody” may also refer to a “naked” antibody fragment.As defined herein, “naked” is synonymous with “unconjugated,” and meansnot linked or conjugated to a therapeutic agent.

Autoimmune Diseases are disorders that are caused by the body producingan immune response against its own tissues. Examples include Class IIIautoimmune diseases such as immune-mediated thrombocytopenias, acuteidiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sjögren's syndrome, multiplesclerosis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, sarcoidosis,ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritisnodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisobliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,rheumatoid arthritis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis, as disclosed in U.S.Provisional Application Ser. No. 60/360,259, filed Mar. 1, 2002, theExamples section of which is incorporated herein by reference.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, the Examples section of eachof which is incorporated herein by reference.

Infectious Diseases as used herein are diseases involving infection bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi,viruses, parasites, or other microbial agents. Examples include humanimmunodeficiency virus (HIV) causing AIDS, Mycobacterium oftuberculosis, Streptococcus agalactiae, methicillin-resistantStaphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes,Escherichia coli, Neisseria gonorrhosae, Neisseria meningitidis,Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum,Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes,West Nile virus, Pseudomonas aeruginosa, Mycobacterium leprae, Brucellaabortus, rabies virus, influenza virus, cytomegalovirus, herpes simplexvirus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, reo virus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodiumvivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi,Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M.pneumoniae. A review listing antibodies against infectious organisms(antitoxin and antiviral antibodies), as well as other targets, iscontained in Casadevall, Clin Immunol 1999; 93(1):5-15, incorporatedherein by reference.

A therapeutic agent is a molecule or atom that is administeredseparately, concurrently or sequentially with a binding moiety, e.g., anantibody or antibody fragment, or a subfragment thereof, and is usefulin the treatment of a disease. Examples of therapeutic agents include,but are not limited to, antibodies, antibody fragments, conjugates,drugs, cytotoxic agents, proapopoptotic agents, toxins, nucleases(including DNAses and RNAses), hormones, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radioisotopes orradionuclides, oligonucleotides, interference RNA, peptides,anti-angiogenic agents, chemotherapeutic agents, cyokines, chemokines,prodrugs, enzymes, binding proteins or peptides or combinations thereof.

A conjugate is an antibody component or other targeting moietyconjugated to a therapeutic agent, such as those described above. Asused herein, the terms “conjugate” and “immunoconjugate” are usedinterchangeably.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked (fused) to another moiety, such as a protein or peptide toxin,cytokine, hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens. An antibody fusion protein comprises at least oneantigen binding site. The valency of the fusion protein indicates thetotal number of binding arms or sites the fusion protein has toantigen(s) or epitope(s); i.e., monovalent, bivalent, trivalent ormutlivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen, or to differentantigens. Specificity indicates how many different types of antigen orepitope an antibody fusion protein is able to bind; i.e., monospecific,bispecific, trispecific, multispecific. Using these definitions, anatural antibody, e.g., an IgG, is bivalent because it has two bindingarms but is monospecific because it binds to one type of antigen orepitope. A monospecific, multivalent fusion protein has more than onebinding site for the same antigen or epitope. For example, amonospecific diabody is a fusion protein with two binding sites reactivewith the same antigen. The fusion protein may comprise a multivalent ormultispecific combination of different antibody components or multiplecopies of the same antibody component. The fusion protein mayadditionally comprise a therapeutic agent.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells, as in therapeutic treatment of autoimmune disease. An example ofan immunomodulator as described herein is a cytokine, which is a solublesmall protein of approximately 5-20 kDa that is released by one cellpopulation (e.g., primed T-lymphocytes) on contact with specificantigens, and which acts as an intercellular mediator between cells. Asthe skilled artisan will understand, examples of cytokines includelymphokines, monokines, interleukins, and several related signalingmolecules, such as tumor necrosis factor (TNF) and interferons.Chemokines are a subset of cytokines. Certain interleukins andinterferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation.

CPT is abbreviation for camptothecin, and as used in the presentapplication CPT represents camptothecin itself or an analog orderivative of camptothecin. The structures of camptothecin and some ofits analogs, with the numbering indicated and the rings labeled withletters A-E, are given in formula 1 in Chart 1 below.

Camptothecin Conjugates

Methods are devised in the following ways for the preparation ofconjugates of chemotherapeutic drugs with targeting moieties (TM), suchas an antibody (MAb). The disclosed methods represent a preferredembodiment of the invention. (1) Solubility of the drug is enhanced byplacing a defined polyethyleneglycol (PEG) moiety (i.e., a PEGcontaining a defined number of monomeric units) between the drug and thetargeting vector, wherein the defined PEG is a low molecular weight PEG,preferably containing 1-30 monomeric units, more preferably containing1-12 monomeric units; (2) a first linker connects the drug at one endand terminates with an acetylene or an azide group at the other end;this first linker comprises a defined PEG moiety with an azide oracetylene group at one end and a different reactive group, such ascarboxylic acid or hydroxyl group, at the other end and saidbifunctional defined PEG is attached to the amine group of an aminoalcohol, and the hydroxyl group of the latter is attached to thehydroxyl group on the drug in the form of a carbonate; alternatively,the non-azide (or acetylene) moiety of said defined bifunctional PEG isoptionally attached to the N-terminus of a L-amino acid or apolypeptide, with the C-terminus attached to the amino group of aminoalcohol, and the hydroxy group of the latter is attached to the hydroxylgroup of the drug in the form of carbonate or carbamate, respectively;(3) a second linker, comprising a targeting moiety-coupling group and areactive group complementary to the azide (or acetylene) group of thefirst linker, namely acetylene (or azide), reacts with the drug-(firstlinker) conjugate via acetylene-azide cycloaddition reaction to furnishthe final bifunctional drug product that is useful for conjugating tothe disease targeting moieties such as disease-targeting antibodies; (4)the antibody-coupling group is designed to be either a thiol or athiol-reactive group; (5) methods are devised for selective regenerationof the 10-hydroxyl group in the presence of the C-20 carbonate inpreparations of drug-linker precursor involving CPT analogs such asSN-38; (6) other protecting groups for reactive hydroxyl groups in drugssuch as the phenolic hydroxyl in SN-38, for example, such ast-butyldimethylsilyl or t-butyldiphenylsilyl are also used, and theseare deprotected by tetrabutylammonium fluoride prior to linking of thederivatized drug to a targeting-vector-coupling moiety; and (6) the10-hydroxyl group of CPT analogs is alternatively protected as an esteror carbonate, other than ‘BOC’, such that the bifunctional CPT isconjugated to a targeting moiety without prior deprotection of thisprotecting group, and the protecting group is readily deprotected underphysiological pH conditions after the bioconjugate is administered. Inthe acetylene-azide coupling, referred to as ‘click chemistry’, theazide part may be on L2 with the acetylene part on L3. Alternatively, L2may contain acetylene, with L3 containing azide. ‘Click chemistry’ is acopper (+1)-catalyzed cycloaddition reaction between an acetylene moietyand an azide moiety, and is a relatively recent technique inbioconjugations (Kolb H C and Sharpless K B, Drug Discov Today 2003; 8:1128-37). Click chemistry takes place in aqueous solution atnear-neutral pH conditions, and is thus amenable for drug conjugation.The advantage of click chemistry is that it is chemoselective, andcomplements other well-known conjugation chemistries such as thethiol-maleimide reaction. In the following discussion, where a conjugatecomprises an antibody or antibody fragment, another type of bindingmoiety, such as an aptamer, avimer or targeting peptide, may besubstituted.

An exemplary preferred embodiment is directed to a conjugate of a drugderivative and an antibody of the general formula 2,MAb-[L2]-[L1]-[AA]_(m)-[A′]-Drug  (2)where MAb is a disease-targeting antibody; L2 is a component of thecross-linker comprising an antibody-coupling moiety and one or more ofacetylene (or azide) groups; L1 comprises a defined PEG with azide (oracetylene) at one end, complementary to the acetylene (or azide) moietyin L2, and a reactive group such as carboxylic acid or hydroxyl group atthe other end; AA is an L-amino acid; m is an integer with values of 0,1, 2, 3, or 4; and A′ is an additional spacer, selected from the groupof ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, orsubstituted or unsubstituted ethylenediamine. The L amino acids of ‘AA’are selected from alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. If the A′ group contains hydroxyl, itis linked to the hydroxyl group or amino group of the drug in the formof a carbonate or carbamate, respectively.

In a preferred embodiment of formula 2, A′ is a substituted ethanolaminederived from an L-amino acid, wherein the carboxylic acid group of theamino acid is replaced by a hydroxymethyl moiety. A′ may be derived fromany one of the following L-amino acids: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine.

In an example of the conjugate of the preferred embodiment of formula 2,m is 0, A′ is L-valinol, and the drug is exemplified by SN-38. Theresultant structure is shown in formula 3.

In another example of the conjugate of the preferred embodiment offormula 2, m is 1 and represented by a derivatized L-lysine, A′ isL-valinol, and the drug is exemplified by SN-38. The structure is shownin formula 4.

In this embodiment, an amide bond is first formed between the carboxylicacid of an amino acid such as lysine and the amino group of valinol,using orthogonal protecting groups for the lysine amino groups. Theprotecting group on the N-terminus of lysine is removed, keeping theprotecting group on the side chain of lysine intact, and the N-terminusis coupled to the carboxyl group on the defined PEG with azide (oracetylene) at the other end. The hydroxyl group of valinol is thenattached to the 20-chloroformate derivative of 10-hydroxy-protectedSN-38, and this intermediate is coupled to an L2 component carrying thetargeting vector-binding moiety as well as the complementary acetylene(or azide) group involved in the click cycloaddition chemistry. Finally,removal of protecting groups at both lysine side chain and SN-38 givesthe product of this example, shown in formula 3.

While not wishing to be bound by theory, the small MW SN-38 product,namely valinol-SN-38 carbonate, generated after intracellularproteolysis, has the additional pathway of liberation of intact SN-38through intramolecular cyclization involving the amino group of valinoland the carbonyl of the carbonate.

In another preferred embodiment, A′ of the general formula 2 is A-OH,whereby A-OH is a collapsible moiety such as 4-aminobenzyl alcohol or asubstituted 4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkyl groupat the benzylic position, and the latter, via its amino group, isattached to an L-amino acid or a polypeptide comprising up to fourL-amino acid moieties; wherein the N-terminus is attached to across-linker terminating in the targeting moiety-binding group.

An example of a preferred embodiment is given below, wherein the A-OHembodiment of A′ of general formula (2) is derived from substituted4-aminobenzyl alcohol, and ‘AA’ is comprised of a single L-amino acidwith m=1 in the general formula (2), and the drug is exemplified withSN-38. The structure is represented below (formula 5, referred to asMAb-CLX-SN-38). Single amino acid of AA is selected from any one of thefollowing L-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. The substituent R on 4-aminobenzylalcohol moiety (A-OH embodiment of A′) is hydrogen or an alkyl groupselected from C1-C10 alkyl groups.

An embodiment of MAb-CLX-SN-38 of formula 5, wherein the single aminoacid AA is L-lysine and R═H, and the drug is exemplified by SN-38(formula 6; referred to as MAb-CL2A-SN-38).

Other embodiments are possible within the context of10-hydroxy-containing camptothecins, such as SN-38. In the example ofSN-38 as the drug, the more reactive 10-hydroxy group of the drug isderivatized leaving the 20-hydroxyl group unaffected. Within the generalformula 2, A′ is a substituted ethylenediamine. An example of thisembodiment is represented by the formula ‘7’ below, wherein the phenolichydroxyl group of SN-38 is derivatized as a carbamate with a substitutedethylenediamine, with the other amine of the diamine derivatized as acarbamate with a 4-aminobenzyl alcohol, and the latter's amino group isattached to Phe-Lys dipeptide. In this structure (formula 7), R and R′are independently hydrogen or methyl. It is referred to asMAb-CL17-SN-38 or MAb-CL2E-SN-38, when R═R′=methyl.

In a preferred embodiment, AA comprises a polypeptide moiety, preferablya di, tri or tetrapeptide, that is cleavable by intracellular peptidase.Examples are: Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu (Trouet et al.,1982).

In another preferred embodiment, the L1 component of the conjugatecontains a defined polyethyleneglycol (PEG) spacer with 1-30 repeatingmonomeric units. In a further preferred embodiment, PEG is a defined PEGwith 1-12 repeating monomeric units. The introduction of PEG may involveusing heterobifunctionalized PEG derivatives which are availablecommercially. The heterobifunctional PEG may contain an azide oracetylene group. An example of a heterobifunctional defined PEGcontaining 8 repeating monomeric units, with ‘NHS’ being succinimidyl,is given below in formula 8:

In a preferred embodiment, L2 has a plurality of acetylene (or azide)groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5,and a single targeting vector-binding moiety.

A representative SN-38 conjugate of an antibody containing multiple drugmolecules and a single targeting vector-binding moiety is shown below.The 12′ component of this structure is appended to 2 acetylenic groups,resulting in the attachment of two azide-appended SN-38 molecules. Thebonding to MAb is represented as a succinimide.

In preferred embodiments, when the bifunctional drug contains athiol-reactive moiety as the antibody-binding group, the thiols on theantibody are generated on the lysine groups of the antibody using athiolating reagent. Methods for introducing thiol groups onto antibodiesby modifications of MAb's lysine groups are well known in the art (Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). Alternatively, mild reduction ofinterchain disulfide bonds on the antibody (Willner et al., BioconjugateChem. 4:521-527 (1993)) using reducing agents such as dithiothreitol(DTT) can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple drug moieties in the interchainregion of the MAb away from the antigen-binding region.

In a preferred embodiment, the preferred chemotherapeutic moiety isselected from the group consisting of doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), CPT, 10-hydroxycamptothecin, SN-38, topotecan, lurtotecan, 9-aminocamptothecin,9-nitrocamptothecin, taxanes, geldanamycin, ansamycins, and epothilones.In a more preferred embodiment, the chemotherapeutic moiety is SN-38.Preferably, in the conjugates of the preferred embodiments, thetargeting moiety links to at least one chemotherapeutic moiety;preferably 1 to about 12 chemotherapeutic moieties; most preferablyabout 6 to about 12 chemotherapeutic moieties.

Furthermore, in a preferred embodiment, the linker component 12′comprises a thiol group that reacts with a thiol-reactive residueintroduced at one or more lysine side chain amino groups of saidtargeting moiety. In such cases, the antibody is pre-derivatized with athiol-reactive group such as a maleimide, vinylsulfone, bromoacetamide,or iodoacetamide by procedures well described in the art.

In the context of these embodiments, a process was surprisinglydiscovered by which CPT drug-linkers can be prepared wherein CPTadditionally has a 10-hydroxyl group. This process involves, but is notlimited to, the protection of the 10-hydroxyl group as at-butyloxycarbonyl (BOC) derivative, followed by the preparation of thepenultimate intermediate of the drug-linker conjugate. Usually, removalof BOC group requires treatment with strong acid such as trifluoroaceticacid (TFA). Under these conditions, the CPT 20-O-linker carbonate,containing protecting groups to be removed, is also susceptible tocleavage, thereby giving rise to unmodified CPT. In fact, the rationalefor using a mildly removable methoxytrityl (MMT) protecting group forthe lysine side chain of the linker molecule, as enunciated in the art,was precisely to avoid this possibility (Walker et al., 2002). It wasdiscovered that selective removal of phenolic BOC protecting group ispossible by carrying out reactions for short durations, optimally 3-to-5minutes. Under these conditions, the predominant product was that inwhich the ‘BOC’ at 10-hydroxyl position was removed, while the carbonateat ‘20’ position was intact.

An alternative approach involves protecting the CPT analog's 10-hydroxyposition with a group other than ‘BOC’, such that the final product isready for conjugation to antibodies without a need for deprotecting the10-OH protecting group. The 10-hydroxy protecting group, which convertsthe 10-OH into a phenolic carbonate or a phenolic ester, is readilydeprotected by physiological pH conditions or by esterases after in vivoadministration of the conjugate. The faster removal of a phenoliccarbonate at the 10 position vs. a tertiary carbonate at the 20 positionof 10-hydroxycamptothecin under physiological condition has beendescribed by He et al. (He et al., Bioorganic & Medicinal Chemistry 12:4003-4008 (2004)). A 10-hydroxy protecting group on SN-38 can be ‘COR’where R can be a substituted alkyl such as “N(CH₃)₂—(CH₂)_(n)—” where nis 1-10 and wherein the terminal amino group is optionally in the formof a quaternary salt for enhanced aqueous solubility, or a simple alkylresidue such as “CH₃—(CH₂)_(n)—” where n is 0-10, or it can be an alkoxymoiety such as “CH₃—(CH₂)n-O—” where n is 0-10, or“N(CH₃)₂—(CH₂)_(n)—O—” where n is 2-10, or“R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl or methyl and n is aninteger with values of 0-10. These 10-hydroxy derivatives are readilyprepared by treatment with the chloroformate of the chosen reagent, ifthe final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-X are as described in earlier sections, thebifunctional drug moiety, [L2]-[L1]-[AA]_(m)-[A-X]-Drug is firstprepared, followed by the conjugation of the bifunctional drug moiety tothe targeting moiety, TM.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-OH are as described in earlier sections,the bifunctional drug moiety is prepared by first linking A-OH to theC-terminus of AA via an amide bond, followed by coupling the amine endof AA to a carboxylic acid group of L1. If AA is absent (i.e m=0), A-OHis directly attached to L1 via an amide bond. The cross-linker,[L1]-[AA]_(m)-[A-OH], is attached to drug's hydroxyl or amino group, andthis is followed by attachment to the L1 moiety, by taking recourse tothe reaction between azide (or acetylene) and acetylene (or azide)groups in L1 and L2 via click chemistry.

In one embodiment, the targeting moiety, TM, is a monoclonal antibody(MAb). In a further embodiment, the targeting moiety may be amultivalent and/or multispecific MAb. The targeting moiety may be amurine, chimeric, humanized, or human monoclonal antibody, and saidantibody may be in intact, fragment (Fab, Fab′, F(ab)₂, F(ab′)₂), orsub-fragment (single-chain constructs) form, or of an IgG1, IgG2a, IgG3,IgG4, IgA isotype, or submolecules therefrom.

In a preferred embodiment, the targeting moiety is a monoclonal antibodythat is reactive with an antigen or epitope of an antigen expressed on acancer or malignant cell. The cancer cell is preferably a cell from ahematopoietic tumor, carcinoma, sarcoma, melanoma or a glial tumor. Apreferred malignancy to be treated according to the present invention isa malignant solid tumor or hematopoietic neoplasm.

In a preferred embodiment, the intracellularly-cleavable moiety may becleaved after it is internalized into the cell upon binding by theMAb-drug conjugate to a receptor thereof, and particularly cleaved byesterases and peptidases.

The targeting moiety is preferably an antibody (including fully human,non-human, humanized, or chimeric antibodies) or an antibody fragment(including enzymatically or recombinantly produced fragments) or bindingproteins incorporating sequences from antibodies or antibody fragments.The antibodies, fragments, and binding proteins may be multivalent andmultispecific or multivalent and monospecific as defined above.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

The skilled artisan will realize that the claimed methods andcompositions may utilize any of a wide variety of antibodies known inthe art. Antibodies of use may be commercially obtained from a widevariety of known sources. For example, a variety of antibody secretinghybridoma lines are available from the American Type Culture Collection(ATCC, Manassas, Va.). A large number of antibodies against variousdisease targets, including but not limited to tumor-associated antigens,have been deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206′ 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art. Isolated antibodies may be conjugated totherapeutic agents, such as camptothecins, using the techniquesdisclosed herein.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (mouse genetically engineered to express humanantibodies) (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXENOMOUSE® (mouse genetically engineered to express human antibodies)and similar animals, the mouse antibody genes have been inactivated andreplaced by functional human antibody genes, while the remainder of themouse immune system remains intact.

The XENOMOUSE® (mouse genetically engineered to express humanantibodies) was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®(mouse genetically engineered to express human antibodies) immunizedwith a target antigen will produce human antibodies by the normal immuneresponse, which may be harvested and/or produced by standard techniquesdiscussed above. A variety of strains of XENOMOUSE® (mouse geneticallyengineered to express human antibodies) are available, each of which iscapable of producing a different class of antibody. Transgenicallyproduced human antibodies have been shown to have therapeutic potential,while retaining the pharmacokinetic properties of normal humanantibodies (Green et al., 1999). The skilled artisan will realize thatthe claimed compositions and methods are not limited to use of theXENOMOUSE® (mouse genetically engineered to express human antibodies)system but may utilize any transgenic animal that has been geneticallyengineered to produce human antibodies.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained, forexample, by pepsin or papain digestion of whole antibodies byconventional methods. For example, antibody fragments may be produced byenzymatic cleavage of antibodies with pepsin to provide a 5S fragmentdenoted F(ab′)₂. This fragment may be further cleaved using a thiolreducing agent and, optionally, a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab fragments and an Fc fragment.Exemplary methods for producing antibody fragments are disclosed in U.S.Pat. No. 4,036,945; U.S. Pat. No. 4,331,647; Nisonoff et al., 1960,Arch. Biochem. Biophys., 89:230; Porter, 1959, Biochem. J., 73:119;Edelman et al., 1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press),and Coligan et al. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (JohnWiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 06, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). In certain embodiments, the techniques and compositionsfor therapeutic agent conjugation disclosed herein may be used withbispecific or multispecific antibodies as the targeting moieties.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of each ofwhich is incorporated herein by reference. Reduction of the peptidelinker length to less than 12 amino acid residues prevents pairing ofV_(H) and V_(L) domains on the same chain and forces pairing of V_(H)and V_(L) domains with complementary domains on other chains, resultingin the formation of functional multimers. Polypeptide chains of V_(H)and V_(L) domains that are joined with linkers between 3 and 12 aminoacid residues form predominantly dimers (termed diabodies). With linkersbetween 0 and 2 amino acid residues, trimers (termed triabody) andtetramers (termed tetrabody) are favored, but the exact patterns ofoligomerization appear to depend on the composition as well as theorientation of V-domains (V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), inaddition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and U.S. Ser. No.11/925,408, the Examples section of each of which incorporated herein byreference). The technique utilizes complementary protein bindingdomains, referred to as anchoring domains (AD) and dimerization anddocking domains (DDD), which bind to each other and allow the assemblyof complex structures, ranging from dimers, trimers, tetramers,quintamers and hexamers. These form stable complexes in high yieldwithout requirement for extensive purification. The DNL technique allowsthe assembly of monospecific, bispecific or multispecific antibodies.Any of the techniques known in the art for making bispecific ormultispecific antibodies may be utilized in the practice of thepresently claimed methods.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens. Some ofthe more preferred target combinations include theose listed in Table 1.This is a list of examples of preferred combinations, but is notintended to be exhaustive.

TABLE 1 Some Examples of multispecific antibodies. First target Secondtarget MIF A second proinflammatory effector cytokine, especiallyHMGB-1, TNF-α, IL-1, or IL-6 MIF Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP-1A, or MIP-1B MIF Proinflammatory effectorreceptor, especially IL-6R, IL- 13R, and IL-15R MIF Coagulation factor,especially TF or thrombin MIF Complement factor, especially C3, C5, C3a,or C5a MIF Complement regulatory protein, especially CD46, CD55, CD59,and mCRP MIF Cancer associated antigen or receptor HMGB-1 A secondproinflammatory effector cytokine, especially MIF, TNF-α, IL-1, or IL-6HMGB-1 Proinflammatory effector chemokine, especially MCP-1, RANTES,MIP-1A, or MIP-1B HMGB-1 Proinflammatory effector receptor especiallyMCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1 Coagulation factor, especiallyTF or thrombin HMGB-1 Complement factor, especially C3, C5, C3a, or C5aHMGB-1 Complement regulatory protein, especially CD46, CD55, CD59, andmCRP HMGB-1 Cancer associated antigen or receptor TNF-α A secondproinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 TNF-α Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP-1A, or MIP-1B TNF-α Proinflammatory effector receptor,especially IL-6R IL- 13R, and IL-15R TNF-α Coagulation factor,especially TF or thrombin TNF-α Complement factor, especially C3, C5,C3a, or C5a TNF-α Complement regulatory protein, especially CD46, CD55,CD59, and mCRP TNF-α Cancer associated antigen or receptor LPSProinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 LPS Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP-1A, or MIP-1B LPS Proinflammatory effector receptor,especially IL-6R IL- 13R, and IL-15R LPS Coagulation factor, especiallyTF or thrombin LPS Complement factor, especially C3, C5, C3a, or C5a LPSComplement regulatory protein, especially CD46, CD55, CD59, and mCRP TFor thrombin Proinflammatory effector cytokine, especially MIF, HMGB-1,TNF-α, IL-1, or IL-6 TF or thrombin Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP-1A, or MIP-1B TF or thrombinProinflammatory effector receptor, especially IL-6R IL- 13R, and IL-15RTF or thrombin Complement factor, especially C3, C5, C3a, or C5a TF orthrombin Complement regulatory protein, especially CD46, CD55, CD59, andmCRP TF or thrombin Cancer associated antigen or receptor

Still other combinations, such as are preferred for cancer therapies,include CD20+CD22 antibodies, CD74+CD20 antibodies, CD74+CD22antibodies, CEACAM5 (CEA)+CEACAM6 (NCA) antibodies, insulin-like growthfactor (ILGF)+CEACAM5 antibodies, EGP-1 (e.g., RS-7)+ILGF antibodies,CEACAM5+EGFR antibodies. Such antibodies need not only be used incombination, but can be combined as fusion proteins of various forms,such as IgG, Fab, scFv, and the like, as described in U.S. Pat. Nos.6,083,477; 6,183,744 and 6,962,702 and U.S. Patent ApplicationPublication Nos. 20030124058; 20030219433; 20040001825; 20040202666;20040219156; 20040219203; 20040235065; 20050002945; 20050014207;20050025709; 20050079184; 20050169926; 20050175582; 20050249738;20060014245 and 20060034759, the Examples section of each incorporatedherein by reference.

Target Antigens and Exemplary Antibodies

In a preferred embodiment, antibodies are used that recognize or bind tomarkers or tumor-associated antigens that are expressed at high levelson target cells and that are expressed predominantly or only on diseasedcells versus normal tissues, and antibodies that internalize rapidly.Antibodies useful within the scope of the present invention include MAbswith properties as described above (and show distinguishing propertiesof different levels of internalization into cells and microorganisms),and contemplate the use of, but are not limited to, in cancer, thefollowing MAbs: LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7(anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb) and hL243 (anti-HLA-DR). Such antibodies are known in the art(e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;7,300,655; 7,312,318; and U.S. Patent Application Publ. No. 20040185053;20040202666; 20050271671; 20060193865; 20060210475; 20070087001; theExamples section of each incorporated herein by reference.) Specificknown antibodies of use include hPAM4 (U.S. Pat. No. 7,282,567), hA20(U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S.Pat. No. 7,300,655), hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat.No. 7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat. No.7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No.7,541,440), hR1 (U.S. Provisional Patent Application 61/145,896), hRS7(U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541,440),AB-PG1-XG1-026 (U.S. patent application Ser. No. 11/983,372, depositedas ATCC PTA-4405 and PTA-4406) and D2/B (WO 2009/130575) the text ofeach recited patent or application is incorporated herein by referencewith respect to the Figures and Examples sections.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD S, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6,alpha-fetoprotein (AFP), VEGF (e.g. bevacizumab, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1, EGP-2 (e.g., 17-1A), EGFreceptor (ErbB1) (e.g., cetuximab), ErbB2, ErbB3, Factor H, FHL-1,Flt-3, folate receptor, Ga 733, GROB, HMGB-1, hypoxia inducible factor(HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF), IFN-γ,IFN-α, IFN-β, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2,IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia,HM1.24, gangliosides, HCG, the HLA-DR antigen to which L243 binds, CD66antigens, i.e., CD66a-d or a combination thereof, MAGE, mCRP, MCP-1,MIP-1A, MIP-1B, macrophage migration-inhibitory factor (MIF), MUC1,MUC2, MUC3, MUC4, MUC5, placental growth factor (PlGF), PSA(prostate-specific antigen), PSMA, PAM4 antigen, NCA-95, NCA-90, A3,A33, Ep-CAM, KS-1, Le(y), mesothelin, 5100, tenascin, TAC, Tn antigen,Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, TNF-α, TRAIL receptor (R1 and R2), VEGFR, RANTES,T101, as well as cancer stem cell antigens, complement factors C3, C3a,C3b, C5a, C5, and an oncogene product.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623), all incorporated in their entirety by reference. Anumber of the aforementioned antigens are disclosed in U.S. ProvisionalApplication Ser. No. 60/426,379, entitled “Use of Multi-specific,Non-covalent Complexes for Targeted Delivery of Therapeutics,” filedNov. 15, 2002, incorporated herein by reference. Cancer stem cells,which are ascribed to be more therapy-resistant precursor malignantcells populations (Gan, J Cell Mol. Med. 2007 Dec. 5 [Epub ahead ofprint]; Hill and Perris, J. Natl. Cancer Inst. 2007; 99(19:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

In multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2007; Oct. 9 (epub aheadof print), and CD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

A recent comprehensive analysis of suitable antigen (ClusterDesignation, or CD) targets on hematopoietic malignant cells, as shownby flow cytometry and which can be a guide to selecting suitableantibodies for drug-conjugated immunotherapy, is Craig and Foon, Bloodprepublished online Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535,incorporated herein by reference.

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)), as well as certain autoimmunediseases. A review of the use of CD74 antibodies in cancer is containedin Stein et al., Clin Cancer Res. 2007 Sep. 15; 13(18 Pt 2):5556s-5563s,incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any chemotherapeutic moiety it carries. Thisallows a high, and therapeutic, concentration of LL1-chemotherapeuticdrug conjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug conjugates are cycled through lysosomes andendosomes, and the chemotherapeutic moiety is released in an active formwithin the target cells.

Dock-and-Lock (DNL)

In certain preferred embodiments, bispecific or multispecific antibodiesmay be produced using the dock-and-lock technology (see, e.g., U.S. Pat.Nos. 7,521,056; 7,550,143; 7,534,866; 7,527,787 and U.S. patentapplication Ser. No. 11/925,408; the Examples section of each of whichis incorporated herein by reference). The DNL method exploits specificprotein/protein interactions that occur between the regulatory (R)subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain(AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). PKA, which plays a central role in one of the beststudied signal transduction pathways triggered by the binding of thesecond messenger cAMP to the R subunits, was first isolated from rabbitskeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763).The structure of the holoenzyme consists of two catalytic subunits heldin an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RIand RII), and each type has α and β isoforms (Scott, Pharmacol. Ther.1991; 50:123). The R subunits have been isolated only as stable dimersand the dimerization domain has been shown to consist of the first 44amino-terminal residues (Newlon et al., Nat. Struct. Biol. 1999; 6:222).Binding of cAMP to the R subunits leads to the release of activecatalytic subunits for a broad spectrum of serine/threonine kinaseactivities, which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). Interestingly, AKAPs will only bind to dimeric Rsubunits. For human RIIα, the AD binds to a hydrophobic surface formedby the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol.1999; 6:216). Thus, the dimerization domain and AKAP binding domain ofhuman RIIα are both located within the same N-terminal 44 amino acidsequence (Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al.,EMBO J. 2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human RIIαand the AD of a certain amino acid sequence as an excellent pair oflinker modules for docking any two entities, referred to hereafter as Aand B, into a noncovalent complex, which could be further locked into astably tethered structure through the introduction of cysteine residuesinto both the DDD and AD at strategic positions to facilitate theformation of disulfide bonds. The general methodology of the“dock-and-lock” approach is as follows. Entity A is constructed bylinking a DDD sequence to a precursor of A, resulting in a firstcomponent hereafter referred to as a. Because the DDD sequence wouldeffect the spontaneous formation of a dimer, A would thus be composed ofa₂. Entity B is constructed by linking an AD sequence to a precursor ofB, resulting in a second component hereafter referred to as b. Thedimeric motif of DDD contained in a₂ will create a docking site forbinding to the AD sequence contained in b, thus facilitating a readyassociation of a₂ and b to form a binary, trimeric complex composed ofa₂b. This binding event is made irreversible with a subsequent reactionto covalently secure the two entities via disulfide bridges, whichoccurs very efficiently based on the principle of effective localconcentration because the initial binding interactions should bring thereactive thiol groups placed onto both the DDD and AD into proximity(Chimura et al., Proc. Natl. Acad. Sci. USA. 2001; 98:8480) to ligatesite-specifically.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors, virtually any protein or peptide may beincorporated into a DNL construct. However, the technique is notlimiting and other methods of conjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide, such as an antibody orfragment. However, the skilled artisan will realize that the site ofattachment of an AD or DDD moiety to an effector moiety may vary,depending on the chemical nature of the effector moiety and the part(s)of the effector moiety involved in its physiological activity.Site-specific attachment of a variety of effector moieties may beperformed using techniques known in the art, such as the use of bivalentcross-linking reagents and/or other chemical conjugation techniques.

In a preferred embodiment, the fusion proteins are assembled by the dockand lock (DNL) techniques disclosed in, e.g., Rossi E A, et al., ProcNatl Acad Sci USA 2006; 103:6841-6846; U.S. Pat. Nos. 7,521,056;7,550,143; 7,534,866; 7,527,787 and U.S. patent application Ser. No.11/925,408; the Examples section of each of which is incorporated hereinby reference. Exemplary DDD and AD sequences that may be utilized in theDNL method to form synthetic complexes are disclosed below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

DNL Sequence Variants

In alternative embodiments, sequence variants of the AD and/or DDDmoieties may be utilized in construction of the DNL complexes. Thestructure-function relationships of the AD and DDD domains have been thesubject of investigation. (See, e.g., Burns-Hamuro et al., 2005, ProteinSci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38; Alto etal., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al., 2006,Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Goldet al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:1below. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding.

Human DDD sequence from protein kinase A (SEQ ID NO: 1)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:5), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:3.

AKAP-IS SEQUENCE (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO:5), exhibiting a five orderof magnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, that increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:6-8. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AKAP-IS sequence (SEQ ID NO:3), theAD moiety may also include the additional N-terminal residues cysteineand glycine and C-terminal residues glycine and cysteine, as shown inSEQ ID NO:4.

SuperAKAP-IS (SEQ ID NO: 5) QIEYVAKQIVDYAIHQA Alternative AKAP sequences(SEQ ID NO: 6) QIEYKAKQIVDHAIHQA (SEQ ID NO: 7) QIEYHAKQIVDHAIHQA(SEQ ID NO: 8) QIEYVAKQIVDHAIHQA

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:9-11. The peptide antagonists were designatedas Ht31 (SEQ ID NO:9), RIAD (SEQ ID NO:10) and PV-38 (SEQ ID NO:11). TheHt-31 peptide exhibited a greater affinity for the RII isoform of PKA,while the RIAD and PV-38 showed higher affinity for RI.

Ht31 (SEQ ID NO: 9) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 10)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 11) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence. The residues are the same asobserved by Alto et al. (2003), with the addition of the C-terminalalanine residue. (See FIG. 4 of Hundsrucker et al. (2006), incorporatedherein by reference.) The sequences of peptide antagonists withparticularly high affinities for the RII DDD sequence are shown in SEQID NO:12-14.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AKAP7δ-wt-pep (SEQ ID NO: 12)PEDAELVRLSKRLVENAVLKAVQQY AKAP7δ-L304T-pep (SEQ ID NO: 13)PEDAELVRTSKRLVENAVLKAVQQY AKAP7δ-L308D-pep (SEQ ID NO: 14)PEDAELVRLSKRDVENAVLKAVQQY

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:1. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A 

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Avimers

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220.) The resultingmultidomain proteins may comprise multiple independent binding domains,that may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, DDD and/orAD sequences for use in the claimed methods and compositions. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756, 20050048512, 20050053973, 20050089932 and 20050221384, theExamples section of each of which is incorporated herein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods forpreparing a phage library. The phage display technique involvesgenetically manipulating bacteriophage so that small peptides can beexpressed on their surface (Smith and Scott, 1985, Science228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. 21:228-257). Inaddition to peptides, larger protein domains such as single-chainantibodies may also be displayed on the surface of phage particles (Arapet al., 1998, Science 279:377-380).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, theExamples section of each incorporated herein by reference. Methods forpreparation and screening of aptamers that bind to particular targets ofinterest are well known, for example U.S. Pat. No. 5,475,096 and U.S.Pat. No. 5,270,163, the Examples section of each incorporated herein byreference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

Conjugation Protocols

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that are facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering a therapeutically effective amount ofa therapeutic conjugate as described herein to a subject. Diseases thatmay be treated with the therapeutic conjugates described herein include,but are not limited to B-cell malignancies (e.g., non-Hodgkin's lymphomaand chronic lymphocytic leukemia using, for example LL2 MAb; see U.S.Pat. No. 6,183,744), adenocarcinomas of endodermally-derived digestivesystem epithelia, cancers such as breast cancer and non-small cell lungcancer, and other carcinomas, sarcomas, glial tumors, myeloid leukemias,etc. In particular, antibodies against an antigen, e.g., an oncofetalantigen, produced by or associated with a malignant solid tumor orhematopoietic neoplasm, e.g., a gastrointestinal, lung, breast,prostate, ovarian, testicular, brain or lymphatic tumor, a sarcoma or amelanoma, are advantageously used. Such therapeutics can be given onceor repeatedly, depending on the disease state and tolerability of theconjugate, and can also be used optimally in combination with othertherapeutic modalities, such as surgery, external radiation,radioimmunotherapy, immunotherapy, chemotherapy, antisense therapy,interference RNA therapy, gene therapy, and the like. Each combinationwill be adapted to the tumor type, stage, patient condition and priortherapy, and other factors considered by the managing physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term.

In a preferred embodiment, therapeutic conjugates comprising the Mu-9MAb can be used to treat colorectal, as well as pancreatic and ovariancancers as disclosed in U.S. Pat. Nos. 6,962,702 and 7,387,772, theExamples section of each incorporated herein by reference. In addition,therapeutic conjugates comprising the PAM4 MAb can be used to treatpancreatic cancer, as disclosed in U.S. Pat. Nos. 7,238,786 and7,282,567, the Examples section of each incorporated herein byreference.

In another preferred embodiment, therapeutic conjugates comprising theRS7 MAb (binding to epithelial glycoprotein-1 [EGP-1] antigen) can beused to treat carcinomas such as carcinomas of the lung, stomach,urinary bladder, breast, ovary, uterus, and prostate, as disclosed inU.S. Pat. No. 7,238,785, the Examples section of which is incorporatedherein by reference.

In another preferred embodiment, therapeutic conjugates comprising theanti-AFP MAb can be used to treat hepatocellular carcinoma, germ celltumors, and other AFP-producing tumors using humanized, chimeric andhuman antibody forms, as disclosed in U.S. Pat. No. 7,300,655, theExamples section of which is incorporated herein by reference.

In another preferred embodiment, therapeutic conjugates comprisinganti-tenascin antibodies can be used to treat hematopoietic and solidtumors and conjugates comprising antibodies to tenascin can be used totreat solid tumors, preferably brain cancers like glioblastomas.

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy.

In another preferred embodiment, the therapeutic conjugates can be usedagainst pathogens, since antibodies against pathogens are known. Forexample, antibodies and antibody fragments which specifically bindmarkers produced by or associated with infectious lesions, includingviral, bacterial, fungal and parasitic infections, for example caused bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi, andviruses, and antigens and products associated with such microorganismshave been disclosed, inter alia, in Hansen et al., U.S. Pat. No.3,927,193 and Goldenberg U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544,4,468,457, 4,444,744, 4,818,709 and 4,624,846, the Examples section ofeach incorporated herein by reference, and in Reichert and Dewitz, citedabove. In a preferred embodiment, the pathogens are selected from thegroup consisting of HIV virus, Mycobacterium tuberculosis, Streptococcusagalactiae, methicillin-resistant Staphylococcus aureus, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhosae, Neisseria meningitidis, Pneumococcus, Cryptococcusneoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus,cytomegalovirus, herpes simplex virus I, herpes simplex virus II, humanserum parvo-like virus, respiratory syncytial virus, varicella-zostervirus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus,human T-cell leukemia viruses, Epstein-Barr virus, murine leukemiavirus, mumps virus, vesicular stomatitis virus, sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus40, mouse mammary tumor virus, dengue virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae, as disclosed inU.S. Pat. No. 6,440,416, the Examples section of which is incorporatedherein by reference.

In a more preferred embodiment, drug conjugates of the present inventioncomprising anti-gp120 and other such anti-HIV antibodies can be used astherapeutics for HIV in AIDS patients; and drug conjugates of antibodiesto Mycobacterium tuberculosis are suitable as therapeutics fordrug-refractive tuberculosis. Fusion proteins of anti-gp120 MAb (antiHIV MAb) and a toxin, such as Pseudomonas exotoxin, have been examinedfor antiviral properties (Van Oigen et al., J Drug Target, 5:75-91,1998). Attempts at treating HIV infection in AIDS patients failed,possibly due to insufficient efficacy or unacceptable host toxicity. Thedrug conjugates of the present invention advantageously lack such toxicside effects of protein toxins, and are therefore advantageously used intreating HIV infection in AIDS patients. These drug conjugates can begiven alone or in combination with other antibiotics or therapeuticagents that are effective in such patients when given alone. Candidateanti-HIV antibodies include the anti-envelope antibody described byJohansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), as well as theanti-HIV antibodies described and sold by Polymun (Vienna, Austria),also described in U.S. Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agens Chemother. 2006; 50(5):1773-9, all incorporated herein byreference. A preferred targeting agent for HIV is various combinationsof these antibodies in order to overcome resistance.

In another preferred embodiment, diseases that may be treated using thetherapeutic conjugates of the preferred embodiments of the presentinvention include, but are not limited to immune dysregulation diseaseand related autoimmune diseases, including Class III autoimmune diseasessuch as immune-mediated thrombocytopenias, such as acute idiopathicthrombocytopenic purpura and chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sjögren's syndrome, multiple sclerosis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, sarcoidosis, ulcerativecolitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa,ankylosing spondylitis, Goodpasture's syndrome, thromboangitisobliterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,rheumatoid arthritis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis, and also juvenile diabetes,as disclosed in U.S. Provisional Application Ser. No. 60/360,259, filedMar. 1, 2002 (now expired). Typical antibodies useful in these diseasesinclude, but are not limited to, those reactive with HLA-DR antigens,B-cell and plasma-cell antigens (e.g., CD19, CD20, CD21, CD22, CD23,CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33,CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7,MUC1, Ia, HM1.24, and HLA-DR), IL-6, IL-17. Since many of theseautoimmune diseases are affected by autoantibodies made by aberrantB-cell populations, depletion of these B-cells by therapeutic conjugatesinvolving such antibodies-therapeutic agent conjugates described hereinis a preferred method of autoimmune disease therapy, especially whenB-cell antibodies are combined, in certain circumstances, with HLA-DRantibodies and/or T-cell antibodies (including those which target IL-2as an antigen, such as anti-TAC antibody). In a preferred embodiment,the anti-B-cell, anti-T-cell, or anti-macrophage or other suchantibodies of use in the treatment of patients with autoimmune diseasesalso can be conjugated to result in more effective therapeutics tocontrol the host responses involved in said autoimmune diseases, and canbe given alone or in combination with other therapeutic agents, such asTNF inhibitors or TNF antibodies, unconjugated B- or T-cell antibodies,and the like.

In a preferred embodiment, a more effective incorporation into cells andpathogens can be accomplished by using multivalent, multispecific ormultivalent, monospecific antibodies. Examples of such bivalent andbispecific antibodies are found in U.S. Pat. Nos. 7,387,772; 7,300,655;7,238,785; and 7,282,567, the Examples section of each of which isincorporated herein by reference. These multivalent or multispecificantibodies are particularly preferred in the targeting of cancers andinfectious organisms (pathogens), which express multiple antigen targetsand even multiple epitopes of the same antigen target, but which oftenevade antibody targeting and sufficient binding for immunotherapybecause of insufficient expression or availability of a single antigentarget on the cell or pathogen. By targeting multiple antigens orepitopes, said antibodies show a higher binding and residence time onthe target, thus affording a higher saturation with the drug beingtargeted in this invention.

In another preferred embodiment, a therapeutic agent used in combinationwith the camptothecin conjugate of this invention may comprise one ormore isotopes. Radioactive isotopes useful for treating diseased tissueinclude, but are not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclide preferably has adecay-energy in the range of 20 to 6,000 keV, preferably in the ranges60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,and 4,000-6,000 keV for an alpha emitter. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or conjugate. Macrocyclicchelates such as NOTA, DOTA, and TETA are of use with a variety ofmetals and radiometals, most particularly with radionuclides of gallium,yttrium and copper, respectively. Such metal-chelate complexes can bemade very stable by tailoring the ring size to the metal of interest.Other ring-type chelates, such as macrocyclic polyethers for complexing²²³Ra, may be used.

Therapeutic agents of use in combination with the camptothecinconjugates described herein also include, for example, chemotherapeuticdrugs such as vinca alkaloids, anthracyclines, epidophyllotoxins,taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2inhibitors, antimitotics, antiangiogenic and proapoptotic agents,particularly doxorubicin, methotrexate, taxol, other camptothecins, andothers from these and other classes of anticancer agents, and the like.Other cancer chemotherapeutic drugs include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidineanalogs, purine analogs, platinum coordination complexes, hormones, andthe like. Suitable chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985), as well as revised editions of thesepublications. Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,aplidin, azaribine, anastrozole, anthracyclines, bendamustine,bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide,estramustine, epidophyllotoxin, estrogen receptor binding agents,etoposide (VP16), etoposide glucuronide, etoposide phosphate,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, gemcitabine,hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide,leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, navelbine, nitrosurea, plicomycin, procarbazine, paclitaxel,pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen,taxol, temazolomide (an aqueous form of DTIC), transplatinum,thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracilmustard, vinorelbine, vinblastine, vincristine and vinca alkaloids. Suchagents may part of the conjugates described herein or may alternativelybe administered in combination with the described conjugates, eitherprior to, simultaneously with or after the conjugate. Alternatively, oneor more therapeutic naked antibodies as are known in the art may be usedin combination with the described conjugates. Exemplary therapeuticnaked antibodies are described in the preceding section.

Therapeutic agents that may be used in concert with the camptothecinconjugates also may comprise toxins conjugated to targeting moieties.Toxins that may be used in this regard include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641,and Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-43.) Additional toxins suitable for use herein are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, a hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β or -γ, and stem cell growth factor, such asthat designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. As used herein, the term cytokine includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, rectal, transmucosal, intestinaladministration, intramuscular, subcutaneous, intramedullary,intrathecal, direct intraventricular, intravenous, intravitreal,intraperitoneal, intranasal, or intraocular injections. The preferredroutes of administration are parenteral. Alternatively, one mayadminister the compound in a local rather than systemic manner, forexample, via injection of the compound directly into a solid tumor.

Immunoconjugates can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

The immunoconjugate can be formulated for intravenous administrationvia, for example, bolus injection or continuous infusion. Preferably,the antibody of the present invention is infused over a period of lessthan about 4 hours, and more preferably, over a period of less thanabout 3 hours. For example, the first 25-50 mg could be infused within30 minutes, preferably even 15 min, and the remainder infused over thenext 2-3 hrs. Formulations for injection can be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate from such a matrix depends upon the molecular weightof the immunoconjugate, the amount of immunoconjugate within the matrix,and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

Generally, the dosage of an administered immunoconjugate for humans willvary depending upon such factors as the patient's age, weight, height,sex, general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of immunoconjugate thatis in the range of from about 1 mg/kg to 25 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. A dosage of 1-20 mg/kg for a 70kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-mpatient. The dosage may be repeated as needed, for example, once perweek for 4-10 weeks, once per week for 8 weeks, or once per week for 4weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy.

Alternatively, an immunoconjugate may be administered as one dosageevery 2 or 3 weeks, repeated for a total of at least 3 dosages. Or,twice per week for 4-6 weeks. If the dosage is lowered to approximately200-300 mg/m² (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a70 kg patient), it may be administered once or even twice weekly for 4to 10 weeks. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months. It has been determined, however, thateven higher doses, such as 20 mg/kg once weekly or once every 2-3 weekscan be administered by slow i.v. infusion, for repeated dosing cycles.The dosing schedule can optionally be repeated at other intervals anddosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

In preferred embodiments, the immunoconjugates are of use for therapy ofcancer. Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastomamultiforme, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors,medullary thyroid cancer, differentiated thyroid carcinoma, breastcancer, ovarian cancer, colon cancer, rectal cancer, endometrial canceror uterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, aswell as head-and-neck cancer. The term “cancer” includes primarymalignant cells or tumors (e.g., those whose cells have not migrated tosites in the subject's body other than the site of the originalmalignancy or tumor) and secondary malignant cells or tumors (e.g.,those arising from metastasis, the migration of malignant cells or tumorcells to secondary sites that are different from the site of theoriginal tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one conjugated antibody or other targeting moiety as describedherein. If the composition containing components for administration isnot formulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

General

Abbreviations used below are: DCC, dicyclohexylcarbodiimide; NHS,N-hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; EEDQ,2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; MMT, monomethoxytrityl;PABOH, p-aminobenzyl alcohol; PEG, polyethylene glycol; SMCC,succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; TBAF,tetrabutylammonium fluoride; TBDMS, tert-butyldimethylsilyl chloride.

Chloroformates of hydroxy compounds in the following examples wereprepared using triphosgene and DMAP according to the procedure describedin Moon et al. (J. Medicinal Chem. 51:6916-6926, 2008), which isincorporated by reference. Extractive work-up refers to extraction withchloroform, dichloromethane or ethyl acetate, and washing optionallywith saturated bicarbonate, water, and with saturated sodium chloride.Flash chromatography was done using 230-400 mesh silica gel andmethanol-dichloromethane gradient, using up to 15% v/vmethanol-dichloromethane, unless otherwise stated. Reverse phase HPLCwas performed by Method A using a 7.8×300 mm C18 HPLC column, fittedwith a precolumn filter, and using a solvent gradient of 100% solvent Ato 100% solvent B in 10 minutes at a flow rate of 3 mL per minute andmaintaining at 100% solvent B at a flow rate of 4.5 mL per minute for 5or 10 minutes; or by Method B using a 4.6×30 mm Xbridge C18, 2.5 μm,column, fitted with a precolumn filter, using the solvent gradient of100% solvent A to 100% of solvent B at a flow rate of 1.5 mL per minutesfor 4 min and 100% of solvent B at a flow rate of 2 mL per minutes for 1minutes. Solvent A was 0.3% aqueous ammonium acetate, pH 4.46 whilesolvent B was 9:1 acetonitrile-aqueous ammonium acetate (0.3%), pH 4.46.HPLC was monitored by a dual in-line absorbance detector set at 360 nmand 254 nm.

Example 1: Preparation of CL6-SN-38

CL6-SN-38 is represented in Scheme-1. Commercially availableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N₃’; 227 mg) was activated with DCC (100 mg), NHS (56 mg), and acatalytic amount of DMAP in 10 mL of dichloromethane for 10 min. To thismixture was added L-valinol (46.3 mg), and the reaction mixture wasstirred for 1 h at ambient temperature. Filtration, followed by solventremoval and flash chromatography yielded 214 mg of clear oily material.This intermediate (160 mg) was reacted with10-O-BOC-SN-38-20-O-chloroformate, the latter generated from10-O-BOC-SN-38 (123 mg) using triphosgene and DMAP. The couplingreaction was done in 4 mL of dichloromethane for 10 min, and thereaction mixture was purified by flash chromatography to obtain 130 mg(45% yield) of product as foamy material. HPLC: t_(R) 11.80 min;electrospray mass spectrum: M+Na: m/z 1181.

The maleimide-containing acetylenic reagent, namely4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide, requiredfor click cycloaddition, was prepared by reacting 0.107 g of SMCC and0.021 mL of propargylamine (0.018 g; 1.01 equiv.) in dichloromethaneusing 1.1 equiv. of diisopropylethylamine. After 1 h, the solvent wasremoved and the product was purified by flash chromatography to obtain83 mg of the product (colorless powder). Electrospray mass spectrumshowed peaks at m/e 275 (M+H) and a base peak at m/e 192 in the positiveion mode, consistent with the structure calculated for C₁₅H₁₈N₂O₃:275.1390 (M+H), found: 275.1394 (exact mass).

The azido intermediate (126 mg) described above was dissolved in DMSO(1.5 mL) and water (0.4 mL), and reacted with 60 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide and 15 mgof cuprous bromide and stirred for 30 min at ambient temperature. Flashchromatography, after work up of the reaction mixture, furnished 116 mg(75% yield) of the cycloaddition product. HPLC: t_(R) 11.20 min;electrospray mass spectrum: M+H and M+Na at m/z 1433 and 1456,respectively. Finally, deprotection with a mixture of TFA (5 mL),dichloromethane (1 mL), anisole (0.1 mL) and water (0.05 mL), followedby precipitation with ether and subsequent flash chromatography yieldedthe product, CL6-SN-38, as a gummy material. HPLC: t_(R) 9.98 min;electrospray mass spectrum: M+H and M−H (negative ion mode) at m/z 1333and 1356, respectively.

Example 2: Preparation of CL7-SN-38

The synthesis is schematically shown in Scheme-2. L-Valinol (40 mg) wasreacted with commercially available Fmoc-Lys(MMT)-OH (253 mg) and EEDQ(107 mg) in 10 mL of anhydrous dichloromethane at ambient temperature,under argon, for 3 h. Extractive work up followed by flashchromatography furnished the product Fmoc-Lys(MMT)-valinol as a paleyellow liquid (200 mg; ˜70% yield). HPLC: t_(R)14.38 min; electrospraymass spectrum: M+H: m/z 727. This intermediate (200 mg) was deprotectedwith diethylamine (10 mL), and the product (135 mg) was obtained in ˜90%purity after flash chromatography. HPLC: t_(R) 10.91 min; electrospraymass spectrum: M+Na at m/z 527. This product (135 mg) was coupled withthe commercially availableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(′PEG-N₃′; 150 mg, 1.1 equiv.) in presence of EEDQ (72 mg, 1.1 equiv.)in 10 mL of dichloromethane, and stirred overnight at ambienttemperature. The crude material was purified by flash chromatography toobtain 240 mg of the purified product as a light yellow oil (˜87%yield). HPLC: t_(R) 11.55 min; electrospray mass spectrum: M+H and M+Naat m/z 1041 and 1063, respectively.

This intermediate (240 mg) was reacted with10-O-TBDMS-SN-38-20-O-chloroformate, the latter generated from10-O-TBDMS-SN-38 (122 mg) using triphosgene and DMAP. The couplingreaction was done in 5 mL of dichloromethane for 10 min, and thereaction mixture was purified by flash chromatography to obtain 327 mgof product as pale yellow foam. Electrospray mass spectrum: M+H at m/z1574. The entire product was reacted with 0.25 mmol of TBAF in 10 mL ofdichloromethane for 5 min, and the reaction mixture was diluted to 100mL and washed with brine. Crude product (250 mg) was dissolved in DMSO(2 mL) and water (0.4 mL), and reacted with 114 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) and 30 mg of cuprous bromide and stirred for1 h at ambient temperature. Flash chromatography furnished 150 mg of thepenultimate intermediate. Finally, deprotection of the MMT group with amixture of TFA (0.5 mL) and anisole (0.05 mL) in dichloromethane (5 mL)for 3 min, followed by purification by flash chromatography yielded 69mg of CL7-SN-38 as a gummy material. HPLC: t_(R) 9.60 min; electrospraymass spectrum: M+H and M−H (negative ion mode) at m/z 1461 and 1459,respectively.

Example 3: Preparation of CL6-SN-38-10-O—CO₂Et

The CL6-SN-38 of Example 1 (55.4 mg) was dissolved in dichloromethane (5mL), and reacted with ethylchloroformate (13.1 mg; 11.5 μL) anddiisopropylethylamine (52.5 mg; 71 μL), and stirred for 20 min underargon. The reaction mixture was diluted with 100 mL of dichloromethane,and washed with 100 mL each of 0.1 M HCl, half saturated sodiumbicarbonate and brine, and dried. Flash chromatography, after solventremoval, furnished 59 mg of the title product. HPLC: t_(R) 10.74 min;exact mass: calc. 1404.6457 (M+H) and 1426.6276 (M+Na); found: 1404.6464(M+H) and 1426.6288 (M+Na).

Example 4: Preparation of CL7-SN-38-10-O—CO₂Et

The precursor of CL7-SN-38 of Example 2 (80 mg) was converted to the10-O-chloroformate using the procedure and purification as described inExample 3. Yield: 60 mg. HPLC: t_(R) 12.32 min; electrospray massspectrum: M+H and M−H (negative ion mode) at m/z 1806 and 1804,respectively. Deprotection of this material using dichloroacetic acidand anisole in dichloromethane gave the title product. HPLC: t_(R) 10.37min; electrospray mass spectrum: M+H at m/z 1534.

Example 5: Preparations of CL6-SN-38-10-O-COR and CL7-SN-38-10-O-COR

This Example shows that the 10-OH group of SN-38 is protected as acarbonate or an ester, instead of as ‘BOC’, such that the final productis ready for conjugation to antibodies without need for deprotecting the10-OH protecting group. This group is readily deprotected underphysiological pH conditions after in vivo administration of the proteinconjugate. In these conjugates, ‘R’ can be a substituted alkyl such as(CH₂)_(n)—N(CH₃)₂ where n is 2-10, or a simple alkyl such as (CH₂)n-CH₃where n is 0-10, or it can be an alkoxy moiety such as “CH₃—(CH₂)n-0-”where n is 0-10, or a substituted alkoxy moiety such as such asO—(CH₂)_(n)—N(CH₃)₂ where n is 2-10 and wherein the terminal amino groupis optionally in the form of a quaternary salt for enhanced aqueoussolubility, or “R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl ormethyl and n is an integer with values of 0-10. In the simplest versionof the latter category, R=“—O—(CH₂)₂—OCH₃”. These 10-hydroxy derivativesare readily prepared by treatment with the chloroformate of the chosenreagent, if the final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused. Such derivatizations are conveniently accomplished using advancedintermediates as illustrated for simple ethyl carbonates of Examples 3and 4.

Example 6: Preparation of CL6-paclitaxel

Valinol is coupled to ‘PEG-N3’ of Scheme-1 according to the proceduredescribed in Example 1. The product is reacted with 0.4 molar equivalentof triphosgene, 3.1 molar equivalent of DMAP, in dichloromethane. After5 minutes, the chloroformate so formed is reacted with an equimolaramount of paclitaxel for 15 minutes at ambient temperature. The reactive2′-hydroxyl group of paclitaxel (the side chain secondary hydroxylgroup) reacts with the chloroformate of the cross-linker. The product isisolated by flash chromatography. This intermediate (0.1 mmol) isdissolved in DMSO (1.5 mL) and water (0.4 mL), and reacted with 60 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) and 15 mg of cuprous bromide and stirred for30 min at ambient temperature. Flash chromatography, after work up ofthe reaction mixture, furnishes the bifunctional paclitaxel, namelyCL6-paclitaxel.

Example 7: Preparation of CL7-Paclitaxel

L-Valinol (40 mg) is reacted with commercially availableFmoc-Lys(MMT)-OH, and the product is then reacted withO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N₃’), as described in Example 2. The chloroformate of thisderivative is formed by the method of Example-6, and reacted with anequimolar amount of paclitaxel. The reactive 2′-hydroxyl group ofpaclitaxel (the side chain secondary hydroxyl group) reacts with thechloroformate of the cross-linker. Click cycloaddition, using4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) is then performed in a manner similar to thatdescribed in Example 6, and the product is finally treated withdichloroacetic acid and anisole to effect removal of the ‘MMT’ groupunder mild conditions. This process furnishes CL7-paclitaxel.

Example 8: Preparation of CL6-[Morpholino Doxorubicin]

Valinol is coupled to ‘PEG-N3’ of Scheme-1 according to the proceduredescribed in Example 1. The product is reacted with 0.4 molar equivalentof triphosgene, 3.1 molar equivalent of DMAP, in dichloromethane. After5 minutes, the chloroformate so formed is reacted with an equimolaramount of morpholino doxorubicin for 15 minutes at ambient temperature.The primary hydroxyl group of morpholino doxorubicin reacts with thechloroformate of the cross-linker. The product is isolated by flashchromatography. This intermediate (0.1 mmol) is dissolved in DMSO (1.5mL) and water (0.4 mL), and reacted with 60 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) and 15 mg of cuprous bromide and stirred for30 min at ambient temperature. Flash chromatography, after work up ofthe reaction mixture, furnishes the bifunctional paclitaxel, namelyCL6-[morpholino doxorubicin].

Example 9: Preparation of CL7-[Morpholino Doxorubicin]

L-Valinol (40 mg) is reacted with commercially availableFmoc-Lys(MMT)-OH, and the product is then reacted withO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N₃’), as described in Example 2. The chloroformate of thisderivative is formed by the method of Example-6, and reacted with anequimolar amount of morpholino doxorubicin. The primary hydroxyl groupof morpholino doxorubicin reacts with the chloroformate of thecross-linker. Click cycloaddition, using4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) is then performed in a manner similar to thatdescribed in Example 6, and the product is finally treated withdichloroacetic acid and anisole to effect removal of the ‘MMT’ groupunder mild conditions. This process furnishes CL7-[morpholinodoxorubicin].

Example 10: Preparation of CL2A-SN-38

To the mixture of commercially available Fmoc-Lys(MMT)-OH (0.943 g),p-aminobenzyl alcohol (0.190 g) in methylene choloride (10 mL) was addedEEDQ (0.382 g) at room temperature and stirred for 4 h. Extractive workup followed by flash chromatograph yielded 1.051 g of material as whitefoam. All HPLC analyses were performed by Method B as stated in‘General’ in section 0148. HPLC ret. time: 3.53 min., Electrospray massspectrum showed peaks at m/e 745.8 (M+H) and m/e 780.3 (M+Cl⁻),consistent with structure. This intermediate (0.93 g) was dissolved indiethylamine (10 mL) and stirred for 2 h. After solvent removal, theresidue was washed in hexane to obtain 0.6 g of the intermediate ((2) inScheme-3) as colorless precipitate (91.6% pure by HPLC). HPLC ret. time:2.06 min. Electrospray mass spectrum showed peaks at m/e 523.8 (M+H),m/e 546.2 (M+Na) and m/e 522.5 (M−H).

This crude intermediate (0.565 g) was coupled with commerciallyavailableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N3’, 0.627 g) using EEDQ in methylene chloride (10 mL). Solventremoval and flash chromatography yielded 0.99 g of the product ((3) inScheme-3; light yellow oil; 87% yield). HPLC ret. time: 2.45 min.Electrospray mass spectrum showed peaks at m/e 1061.3 (M+H), m/e 1082.7(M+Na) and m/e 1058.8 (M−H), consistent with structure. Thisintermediate (0.92 g) was reacted with10-O-TBDMS-SN-38-20-O-chloroformate ((5) in Scheme-3) in methylenechloride (10 mL) for 10 min under argon. The mixture was purified byflash chromatography to obtain 0.944 g as light yellow oil ((6) inScheme-3; yield=68%). HPLC ret. time: 4.18 min. To this intermediate(0.94 g) in methylene chloride (10 mL) was added the mixture of TBAF (1Min THF, 0.885 mL) and acetic acid (0.085 mL) in methylene chloride (3mL), then stirred for 10 min. The mixture was diluted with methylenechloride (100 mL), washed with 0.25 M sodium citrate and brine. Thesolvent removal yielded 0.835 g of yellow oily product. HPLC ret. time:2.80 min., (99% purity). Electrospray mass spectrum showed peaks at m/e1478 (M+H), m/e 1500.6 (M+Na), m/e 1476.5 (M−H), m/e 1590.5 (M+TFA),consistent with structure.

This azido-derivatized SN-38 intermediate (0.803 g) was reacted with4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (0.233 g)in methylene chloride (10 mL) in presence of CuBr (0.0083 g,), DIEA(0.01 mL) and triphenylphosphine (0.015 g), for 18 h. Extractive workup, including washing with and 0.1M EDTA (10 mL), and flashchromatography yielded 0.891 g as yellow foam. (yield=93%), HPLC ret.time: 2.60 min. Electrospray mass spectrum showed peaks at m/e 1753.3(M+H), m/e 1751.6 (M−H), 1864.5 (M+TFA), consistent with structure.Finally, deprotection of the penultimate intermediate (0.22 g) with amixture of dichloroacetic acid (0.3 mL) and anisole (0.03 mL) inmethylene chloride (3 mL), followed by precipitation with ether yielded0.18 g (97% yield) of CL2A-SN-38; (7) in Scheme-3) as light yellowpowder. HPLC ret. time: 1.88 min. Electrospray mass spectrum showedpeaks at m/e 1480.7 (M+H), 1478.5 (M−H), consistent with structure.

Example 11: Preparation of CL2E-SN-38

N,N′-dimethylethylenediamine (3 mL) in methylene chloride (50 mL) wasreacted with monomethoxytrityl chloride (1.7 g). After 1 h of stirring,the solvent was removed under reduced pressure, and the crude productwas recovered by extractive work up (yellow oil; 2.13 g). All HPLCanalyses were performed by Method B as stated in ‘General’ in section0148. HPLC ret. time: 2.28 min. This intermediate ((1) in Scheme-4; 0.93g) was added in situ to activated SN-38, and the latter ((2) inScheme-4) was prepared by reacting SN-38 (0.3 g) withp-nitrophenylchloroformate (0.185 g) and DIEA (0.293 mL) in DMF for 1 h.After removing solvent, the residue was purified on deactivated silicagel to obtain 0.442 g as white solid.

This intermediate (0.442 g) was deprotected with a mixture oftrifluoroacetic acid (1 mL) and anisole (0.1 mL) in methylene chloride(5 mL), followed by precipitation with ether to obtain 0.197 g of theproduct ((3) in Scheme-4) as white solid. This intermediate ((3); 0.197g) was coupled with activated azide-containing-dipeptideincorporated-PEG-linker ((5) in Scheme-4), which activation was done byreacting PEG-linker ((4) in Scheme-4; 0.203 g) with bis(4-nitrophenyl)carbonate (0.153 g) and DIEA (0.044 mL) in methylene chloride (8 mL).Flash chromatography yielded 0.2 g of azide-derivatized SN-38intermediate product ((6) in Scheme-4) as glassy solid. HPLC ret. time:2.8 min. Electrospray mass spectrum showed peaks at m/e 1740.5 (M+H),m/e 1762.9 (M+Na), m/e 1774.9 (M+Cl⁻), consistent with structure. Thisintermediate ((6) in Scheme-4; 0.2 g) was subjected to clickcycloaddition with4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (0.067 g)in methylene chloride in presence of CuBr (0.007 g,), DIEA (0.008 mL)and triphenylphosphine (0.012 g) for 18 h. Work up of reaction mixture,which included treatment with 0.1M EDTA, followed by flashchromatography yielded 0.08 g of the penultimate intermediate as lightyellow foam. HPLC: t_(R)=2.63 min. Electrospray mass spectrum showedpeaks at m/e 2035.9 (M+Na⁺), m/e 2047.9 (M+Cl⁻), consistent withstructure. Finally, deprotection of this intermediate (0.08 g) with amixture of trifluoroacetic acid (0.2 mL), anisole (0.12 mL) and water(0.06 mL) in methylene chloride (2 mL), followed by precipitation withether yielded 0.051 g of product, CL17-SN-38 (also referred to asCL2E-SN-38), as light yellow powder (yield=69%). HPLC ret. time: 1.95min., ˜99% purity. Electrospray mass spectrum showed peaks at m/e 1741.1(M+H), 1775.5 (M+Cl⁻), consistent with structure.

Example 12: Conjugation of Bifunctional SN-38 Products to Mildly ReducedAntibodies

The anti-CEACAM5 humanized MAb, hMN-14, the anti-CD22 humanized MAb,hLL2, the anti-CD20 humanized MAb, hA20, the anti-EGP-1 humanized MAb,hRS7, and anti-mucin humanized MAb, hPAM4, were used in these studies.Each antibody was reduced with dithiothreitol (DTT), used in a50-to-70-fold molar excess, in 40 mM PBS, pH 7.4, containing 5.4 mMEDTA, at 37° C. (bath) for 45 min. The reduced product was purified bysize-exclusion chromatography and/or diafiltration, and wasbuffer-exchanged into a suitable buffer at pH 6.5. The thiol content wasdetermined by Ellman's assay, and was in the 6.5-to-8.5 SH/IgG range.Alternatively, the antibodies were reduced withTris(2-carboxyethyl)phosphine (TCEP) in phosphate buffer at pH in therange of 5-7, followed by in situ conjugation. The reduced MAb wasreacted with ˜10-to-15-fold molar excess of ‘CL6-SN-38’ of Example 1, or‘CL7-SN-38’ of Example 2, or ‘CL6-SN-38-10-O—CO₂Et’ of Example 3, or‘CL7-SN-38-10-O—CO₂Et’ of Example 4, CL2A-SN-38 of Example 10, orCL2E-SN-38 of Example 11 using DMSO at 7-15% v/v as co-solvent, andincubating for 20 min at ambient temperature. The conjugate was purifiedby centrifuged SEC, passage through a hydrophobic column, and finally byultrafiltration-diafiltration. The product was assayed for SN-38 byabsorbance at 366 nm and correlating with standard values, while theprotein concentration was deduced from absorbance at 280 nm, correctedfor spillover of SN-38 absorbance at this wavelength. This way, theSN-38/MAb substitution ratios were determined. The purified conjugateswere stored as lyophilized formulations in glass vials, capped undervacuum and stored in a −20° C. freezer. SN-38 molar substitution ratios(MSR) obtained for some of these conjugates, which were typically in the5-to-7 range, are shown in Table 2.

TABLE 2 SN-38/MAb Molar substitution ratios (MSR) in some conjugates MAbConjugate MSR hMN-14 hMN-14-[CL2A-SN-38], using drug-linker of 6.1Example 10 hMN-14-[CL6-SN-38], using drug-linker of 6.8 Example 1hMN-14-[CL7-SN-38], using drug-linker of 5.9 Example 2hMN-14-[CL7-SN-38-10-O—CO₂Et], using 5.8 drug-linker of Example 4hMN-14-[CL2E-SN-38], using drug-linker of 5.9 Example 11 hRS7hRS7-CL2A-SN-38 using drug-linker of Example 10 5.8 hRS7-CL7-SN-38 usingdrug-linker of Example 2 5.9 hRS7-CL7-SN-38 (Et) using drug-linker ofExample 4 6.1 hA20 hA20-CL2A-SN-38 using drug-linker of Example 10 5.8hLL2 hLL2-CL2A-SN-38 using drug-linker of Example 10 5.7 hPAM4hPAM4-CL2A-SN-38 using drug-linker of Example 10 5.9

Example 13: In Vivo Therapeutic Efficacies in Preclinical Models ofHuman Pancreatic or Colon Carcinoma

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. FIG. 1 shows a Capan 1 pancreatic tumor model, whereinspecific CL2A-SN-38 conjugates of hRS7 (anti-EGP-1), hPAM4 (anti-mucin),and hMN-14 (anti-CEACAM5) antibodies showed better efficacies thancontrol hA20-CL2A-SN-38 conjugate (anti-CD20) and untreated control.Similarly in a BXPC3 model of human pancreatic cancer, the specifichRS7-CL2A-SN-38 showed better therapeutic efficacy than controltreatments (FIG. 2). Likewise, in an aggressive LS174T model of humancolon carcinoma, treatment with specific hMN-14-CL2A-SN-38 was moreefficacious than non-treatment (FIG. 3).

Example 14: Elimination of HIV Infection by Treatment with an SN-38Conjugate of an Anti-Gp120 MAb

A MAb targeted to the HIV envelope protein gp120, anti-gp120 antibodysuch as P4/D10, is reduced using conditions described in Example 7, andthe reduced MAb is reacted with a 20-fold molar excess of the druglinker CL7-SN-38, which is as described in Example 2. Ananti-gp120-SN-38 conjugate with a substitution of ˜8 drug molecules perantibody is obtained. An in vitro HIV-inhibition assay with saidconjugate is performed by using various mixtures of uninfected Jurkat-Tcells and fully HIV-infected Jurkat T-cells (in the ratios of 99.8:0.2to 95:5), and treating with serial dilutions of the conjugate,non-specific hRS7-CL7-SN38 conjugate control, naked antibody, andHIV-negative serum from 100 to 0.00001 μg/mL. The cells so treated areincubated in RPMI 1640 culture medium at 37° C. for seven days, and thenassayed for HIV inhibition by ELISA test. This experiment shows a strongand specific inhibition of intercellular spread of HIV by the specificdrug conjugate. The in vivo efficacy is tested by administering to miceisologous HIV-infected cells together with specific and non-specificSN-38 conjugates. For this, primary murine splenocytes infected byHIV-1/MuLV pseudotype virus are intraperitoneally transferred to groupsof mice simultaneously with immunoconjugate administration. Peritonealcells are harvested 10 days later. While infectious HIV presence isdemonstrated in control mice, no infectious HIV is detected in micetreated with 100 μg or less of anti-gp120-SN-38 conjugate. No protectionis seen with mice treated with control conjugates.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

We claim:
 1. A method of treating cancer comprising administering to asubject with cancer an immunoconjugate having a structural formulaMAb-linker-SN-38, wherein the linker-SN-38 has a structure representedby:

wherein n is between 1 and 30; AA is an L-amino acid residue selectedfrom the group consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine; and MAb is an IgG antibodyor antigen-binding antibody fragment.
 2. The method of claim 1, whereinn is between 1 and
 12. 3. The method of claim 1, wherein n is between 6and
 12. 4. The method of claim 1, wherein n is
 8. 5. The method of claim1, wherein AA is a lysine residue.
 6. The method of claim 1, wherein theMAb is a murine, chimeric, humanized, or human monoclonal antibody orantigen binding fragment thereof.
 7. The method of claim 6, wherein saidfragment is selected from the group consisting of Fab, Fab′, F(ab)₂,F(ab′)₂, and scFv.
 8. The method of claim 6, wherein the MAb hasconstant domains and a hinge domain of a human IgG1 or a human IgG4antibody.
 9. The method of claim 6, wherein the MAb has constant domainsand a hinge domain of a human IgG4 antibody, wherein serine 228 of thehinge is replaced with proline.
 10. The method of claim 6, wherein theantibody has constant domains and a hinge domain of a human IgG1antibody and wherein one or more Fc amino acids are mutated to increasethe half-life of the antibody in the blood.
 11. The method of claim 1,wherein said MAb is selected from the group consisting of hLL1, hLL2,RFB4, hA19, hA20, hRS7, hPAM4, KC4, hMN-3, hMN-14, hMN-15, hMu-9, hR1,CC49, hL243, D2/B and hIMMU-31.
 12. The method of claim 1, wherein saidMab binds to a tumor-associated antigen (TAA).
 13. The method of claim12, wherein the TAA is selected from the group consisting of carbonicanhydrase IX, B7, CCL19, CCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2,CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21,CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L,CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a,CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6,alpha-fetoprotein (AFP), VEGF, ED-B fibronectin, EGP-1, EGP-2, EGFreceptor (ErbB1), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor,Ga 733, GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24,HER-2/neu, insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-β,IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8,IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24,gangliosides, HCG, HLA-DR, CD66a-d, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B,macrophage migration-inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4,MUC5ac, placental growth factor (PlGF), PSA (prostate-specific antigen),PSMA, PSMA dimer, PAM4 antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1,Le(y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreichantigens, tumor necrosis antigens, tumor angiogenesis antigens, TNF-α,TRAIL receptor (R1 and R2), VEGFR, RANTES, T101, cancer stem cellantigens, complement factors C3, C3a, C3b, C5a, C5, and an oncogeneproduct.
 14. The method of claim 1, wherein the immunoconjugate is madeby reacting a maleimide moiety of the linker-SN38 with a reducedsulfhydryl of the MAb.
 15. The method of claim 1, wherein theimmunoconjugate is made by: a) generating one or more thiols on theantibody by reacting at least one lysine residue on the antibody with athiolating reagent; and b) reacting a maleimide moiety of thelinker-SN38 with the thiol group on the lysine residue.
 16. The methodof claim 1, wherein said MAb is attached to between 1 and 12 copies ofthe linker-SN38.
 17. The method of claim 1, wherein said MAb is attachedto between 6 and 8 copies of linker-SN38.
 18. The method of claim 1,wherein said MAb is attached to between 1 and 5 copies of linker-SN38.19. The method of claim 1, wherein the linker is CL2A, wherein n is 8and AA is a lysine residue.
 20. A method of treating cancer comprisingadministering to a subject with cancer an immunoconjugate having astructural formula MAb-linker-SN-38, wherein the linker-SN-38 has astructure represented by:

wherein n is between 1 and 30; AA is an L-amino acid residue selectedfrom the group consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine; R is a thiol-reactivemoiety; and MAb is an IgG antibody or antigen-binding antibody fragment.21. The method of claim 20, wherein the thiol-reactive moiety isselected from the group comprised of maleimide, vinylsulfone,bromoacetamide and iodoacetamide.
 22. The method of claim 21, whereinthe maleimide moiety is a trans 4-maleimidomethylcyclohexyl moiety. 23.The method of claim 22, wherein the immunoconjugate is made by reactingthe thiol-reactive moiety of the linker-SN38 with a reduced sulfhydrylof the MAb.
 24. The method of claim 22, wherein the immunoconjugate ismade by: a) generating one or more thiols on the antibody by reacting atleast one lysine residue on the antibody with a thiolating reagent; andb) reacting the thiol-reactive moiety of the linker-SN38 with the thiolgroup on the lysine residue.